In 1935, Stein and Leventhal recognized the association of enlarged polycystic ovaries with amenorrhea, hirsutism and infertility. (Stein and Leventhal, 1935) Since then, the increased ovarian androgen production, hyperandrogenemia and menstrual irregularity have come to be known as polycystic ovary syndrome. (Rosenfeld, et al. 1972) Polycystic ovarian syndrome (PCO) has been described as one of the most common female endocrine disorders. Its incidence has been estimated to be about 5% in both adolescent and adult populations. (Declercq and van de Calseyde, 1977) It is characterized by hyperandrogenic chronic anovulation (increased androgen concentrations and cessation of ovulatory cycles), and clinically presents in the prepubertal period with any of the following: irregular menses, amenorrhea, dysfunctional uterine bleeding and hirsutism. (Frank, 1995) It is the most frequent cause of anovulation with approximately 55% of patients presenting with amenorrhea (absence of menses) and 70% with hirsutism. The syndrome comprises a spectrum of ovarian histological and morphological findings, ovarian steroid, gonadotropin and metabolic abnormalities.
The precise pathophysiologic mechanisms resulting in these endocrinologic disturbances is not known and is under intense debate. The most widely accepted theory is that PCO is a heterogenous disorder that increases intraovarian concentrations of androgens. (Barnes, and Rosenfeld, 1989) What is known, is that, there is a self perpetruating cycle of hormonal events resulting in polycystic ovaries, theca cell hyperplasia and arrested follicular cell development. (McKenna, 1988) Ovarian or adrenal androgens convert primarily to estrone in the periphery and feed back on the hypothalamic-pituitary axis to induce gonadotropin-releasing hormone (GnRH) synthesis with increased luteinizing hormone (LH) secretion and reduced follicular stimulating hormone (FHS) production. Increased LH secretion leads to hyperplasia of the ovarian stroma to produce more testosterone and androstenedione, inhibits production of sex hormone binding globulin and increases free androgen. This, then blocks follicular maturation leading to numerous follicles in various stages of development and eventually atresia. So, that a combination of hyperestronemia with hyperandrogenemia probably provides the hormonal milieu for PCO to occur. Evidence suggests that a dysregulation of the rate limiting enyme, cytochrome P450, resulting from elevations in luteinizing hormone, intrinsic defects of theca cell function or from hyperinsulinemia may then masculinize ovarian follicles causing follicular arrest accelerating atresia, and initiating the syndrome. (Rosenfeld, et al., 1990)
In recent years, PCO has been associated with a characteristic metabolic disturbance, insulin resistance and hyperinsulinemia. (Dunaif, et al., 1989) The hyperinsulinemia in PCO is not seen in all women but is more prevalent in obese young women. Evidence suggests that disordered insulin metabolism may precipitate increased androgen levels, while suppression of insulin levels with diazoxide or metformin can cause resumption of menses. Insulin stimulates androgen secretion in ovarian stroma in vitro and may act on the ovary via insulin growth factor receptors. (Barbieri, et al., 1986; Adashi, et al., 1985) The cellular mechanism underlying insulin resistance may reflect reduced binding of insulin to its receptor or a decreased expression of the insulin dependent glucose transporter protein GLT-4. (Jialal, et al.1987;Rosenbaum, et al., 1993) Treatment of PCO has traditionally been directed toward interrupting the self-perpetuating cycle of hormonal events. This has been done either with the use of surgery as in wedge resection of the ovary or through medical interventions by lowering LH levels (oral contraceptives and LHRH analogues). Other approaches have included enhancement of FSH secretion with clomiphene, human menopausal gonadotrophin or pulsatile LHRH therapy.
Over the past 20 years, numerous studies in invertebrates and vertebrates have established a role of calcium in oocyte maturation as well as in the resumption and progression of follicular development. Attention had originally centered on the significance of calcium during egg activation at fertilization by either sperm or the divalent ionophore A23187. Based on evidence that the calcium ionophore A23187 activated the eggs of vertebrates and marine invertebrates by mediating calcium fluxes, Steinhardt and colleagues proposed in 1974 that calcium may have a universal role in egg activation. (Steinhardt, et al. 1974) The maturation of the immature oocyte, and the activation and fertilization of the mature egg are two separate events in mammals. Evidence now exists that increases in intracellular calcium or calcium transients are clearly important at fertilization for invertebrates and vertebrates alike, and clearly important in the maturation of the non-mammalian oocytes. What is not clearly defined is the role of calcium in the meiotic maturation of the mammalian oocyte. The growth and maturation of the oocyte from primordial germ cell to oogonia to oocyte and then egg involves a series of mitotic and meiotic cell divisions. The transition from one meiotic phase to another is generally regulated at 3 control points and in many species, the control points are triggered by increases in intracellular calcium or by overriding meiotic arrest. (Lindner, et al., 1974) Primordial germ cells differentiate into oogonia with the final mitotic division in the fetal ovary to form a finite number of oocytes. At or following birth, the oocyte enters the dictyate stage (or G2 phase in mitotic cells) with an intact nuclear membrane termed the germinal vesicle. It is at this stage of oocyte development, that meiosis becomes arrested, and does not resume until puberty under the influence of LH. Under the influence of LH, the germinal vesicle breaks down and the oocyte enters M phase with resumption of meiosis and extrusion of the 1st polar body. Meiosis is then arrested again, awaiting the signal for fertilization. What is known is that: 1) denuded oocytes that are devoid of cumulus, spontaneously mature; 2) removal of the oocyte from its inhibitory environment or follicle results in spontaneous maturation, so that the oocyte is all set to go; 3) the immature oocyte, is generally accepted to be under the influence of the follicular environment or cumulus and is maintained in meiotic arrest; 4) increased cyclic AMP produced in the granulosa or cumulus oophorus cells helps to maintain meiotic arrest. What overrides meiotic arrest and stimulates meiotic resumption? LH is well established as the biological trigger in mammalian meiotic resumption. Luteinizing hormone is the physiological signal at puberty for oocyte maturation in both in vivo and in vitro studies. (Whitaker and Patel, 1990; Tsafriri, et al., 1972) Calcium is an important participant in the transduction of numerous signals in various tissues, and evidence suggests that LH release is calcium dependent via gonadotropin-releasing hormone induced sensitization of calcium dependent exocytosis. (Jobin, et al., 1995) Furthermore calcium is believed to be the primary intracellular signal in invertebrates and amphibians for the maturation of the oocyte and for initiation of development of the egg at fertilization. This hypothesis has been supported by three experimental models in different species: 1) an increase in calcium in the egg occurs at fertilization; 2) artificially raising intracellular calcium usually initiates egg development; 3) suppressing the natural rise in calcium prevents the initiation of egg development. LH stimulates rapid increases in intracellular calcium in both cow luteal and pig granulosa cells. (Channing, et al., 1978; Davis, et al., 1987; Mattioli, et al., 1991) These LH mediated calcium oscillations are characterized by an initial transient through intracellular calcium mobilization followed by a second wave resulting from calcium influx from the extracellular environment. Manipulation of extracellular calcium such as high concentrations of calcium can override meiotic arrest in mouse oocytes whereas reduced calcium concentrations can inhibit meiotic resumption of follicle-enclosed rat oocytes following LH stimulation. (Powers and Paleos, 1982) In other words, increases or changes in intracellular calcium are required for the oocyte to mature or progress to the next stage. In mammals, a series of calcium transients occur at fertilization and appear to be required for release of the second metaphase arrest, but a role for calcium in triggering meiotic resumption and progression in mammalian oocyte is still not clearly defined, though it is strongly suspected. (Homa, 1995) Similarly, the role of vitamin D in oocyte maturation has not been defined. Nor has a role for calcium and vitamin D in PCO, hirsutism, acne development and fertility been defined.
What triggers this vicious hormonal cycle with altered feedback loops resulting in arrested follicular development and the clinical entity of PCO, to date, has not been fully explained. Primary abnormalities of the ovary, adrenal and pituitary have been proposed and much debate has focused on whether it is even one disorder or multiple. It is proposed, here, that calcium dysregulation inhibits normal follicular development resulting in infertility, oligo/amenorrhea, acne formation and hirsutism. This invention establishes an essential role of calcium regulation in the polycystic ovarian syndrome in that correction of the calcium abnormalities with calcium, vitamin D or calcium and vitamin D can either reverse or improve symptoms of the syndrome.